Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
DOI 10.1186/s12862-015-0325-1
RESEARCH ARTICLE
Open Access
Intra-genomic variation in symbiotic
dinoflagellates: recent divergence or
recombination between lineages?
Shaun P Wilkinson1, Paul L Fisher1, Madeleine JH van Oppen2 and Simon K Davy1*
Abstract
Background: The symbiosis between corals and the dinoflagellate alga Symbiodinium is essential for the development
and survival of coral reefs. Yet this fragile association is highly vulnerable to environmental disturbance. A coral’s ability
to tolerate temperature stress depends on the fitness of its resident symbionts, whose thermal optima vary extensively
between lineages. However, the in hospite population genetic structure of Symbiodinium is poorly understood and
mostly based on analysis of bulk DNA extracted from thousands to millions of cells. Using quantitative single-cell PCR,
we enumerated DNA polymorphisms in the symbionts of the reef-building coral Pocillopora damicornis, and applied a
model selection approach to explore the potential for recombination between coexisting Symbiodinium populations.
Results: Two distinct Symbiodinium ITS2 sequences (denoted C100 and C109) were retrieved from all P. damicornis
colonies analysed. However, the symbiont assemblage consisted of three distinct Symbiodinium populations: cells
featuring pure arrays of ITS2 type C109, near-homogeneous cells of type C100 (with trace ITS2 copies of type C109),
and those with co-dominant C100 and C109 ITS2 repeats. The symbiont consortia of some colonies consisted almost
entirely of these putative C100 × C109 recombinants.
Conclusions: Our results are consistent with the occurrence of sexual recombination between Symbiodinium
types C100 and C109. While the multiple-copy nature of the ITS2 dictates that the observed pattern of intra-genomic
co-dominance may be a result of incomplete concerted evolution of intra-genomic polymorphisms, this is a less likely
explanation given the occurrence of homogeneous cells of the C109 type. Conclusive evidence for inter-lineage
recombination and introgression in this genus will require either direct observational evidence or a single-cell
genotyping approach targeting multiple, single-copy loci.
Keywords: Coral, Symbiodinium, Symbiosis, Pocillopora damicornis, ITS2, Concerted evolution, Sexual reproduction,
Recombination
Background
The ecological success of scleractinian corals arises from
their mutualistic symbiosis with the dinoflagellate alga
Symbiodinium. Energy-rich compounds provided by the
phototrophic endosymbiont enhance coral growth and
enable reef development in nutrient-poor tropical oceans
(reviewed in [1]). The Symbiodinium genus constitutes a
genetically diverse assemblage [2], with several clades
and sub-clades (types) showing different physiological and
ecological characteristics [3-5]. Of particular relevance,
* Correspondence: [email protected]
1
School of Biological Sciences, Victoria University of Wellington, Kelburn
Parade, Wellington 6012, New Zealand
Full list of author information is available at the end of the article
differences in symbiont thermal optima are conferred to
the host in the form of resistance or susceptibility to coral
bleaching [3,5,6], a condition responsible for several largescale episodes of coral mortality [7-9]. Surviving colonies
may acclimatize to warming conditions by replacing
thermally-sensitive symbionts with more robust types
(‘adaptive bleaching’ or ‘symbiont shuffling’; [3,6,10-14]).
However the modified consortium may be unstable [11]
and less mutualistic [15]. Furthermore, host-symbiont coevolution fosters strong fidelity between symbiotic partners [16] and hence this strategy appears to be confined to
a subset of ‘symbiont flexible’ coral taxa [17]. Finally, symbiont shuffling appears to offer a maximum increase in
thermal tolerance of around 1–1.5°C [6]. Given that recent
© 2015 Wilkinson et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
model simulations predict a 1.5-3°C increase by the year
2050 [18], adaptive bleaching will unlikely mitigate the
environmental stress that corals are expected to face in
the near future. As such, the ‘macro-evolutionary’ potential of the coral symbiosis will likely play a defining
role in determining the future of coral reef ecosystems
in a warming climate.
Adaptation may occur in Symbiodinium through selection acting on both existing genetic variation [19,20] and
new genetic variation arising through somatic mutations
[21] and/or genetic recombination. Symbiodinium is generally considered a predominantly asexual, permanent
haploid lineage that diversifies through host-specialization
and geographic isolation [19]; however several lines of
molecular evidence suggest that cryptic meiotic recombination occurs in this genus. Incongruence between isoenzyme phylogenies and those constructed from both RAPD
[22,23] and ITS2 sequence variation [24] implicate allelic
recombination, consistent with criteria outlined to distinguish between clonal and sexual eukaryote populations
[25]. More recently, meiotic recombination has been inferred from linkage disequilibrium between microsatellite
loci [26-30], indicating that extensive shuffling of alleles
has occurred within several Symbiodinium lineages. Additionally, a recent meta-genomic analysis revealed the
presence of six meiosis-specific and 25 meiosis-related
functional genes in published Symbiodinium genomes
[31], providing further evidence that the loss of sexual
reproduction has not occurred in this genus.
Morphological similarities among symbiotic dinoflagellates highlight the need to use appropriate genetic tools
when addressing the incidence of recombination in this
group. Several population- and ‘species’-level markers
are currently available, including polymorphic microsatellite loci (e.g. [26-29]), low-copy nuclear genes such as
actin (e.g. [32]), and mitochondrial and chloroplast sequences (e.g. [33-35]). Yet each of these has drawbacks,
such as low taxonomic resolution or a lack of universal
primer-binding sequences. The internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA (rDNA)
is currently the most well-characterised and commonlyused marker in Symbiodinium systematics [36]. This is
primarily due to its high taxonomic resolution [37-39] and
ease of PCR amplification (due to high copy numbers and
conserved adjacent sequences). Recent genome-wide pyrosequencing has confirmed the taxonomic utility of the
ITS2 region, with the dominant sequence variant offering
97% discrimination efficiency across a range of taxa, and
rare intra-genomic variants further aiding species identification [40,41]. Emerging patterns of ITS2 secondary structure promise even higher taxonomic resolving power [41],
leading some authors to support its candidacy as a barcode marker for delineating species boundaries in plants
and algae [42,43]. Unlike alternative mitochondrial- and
Page 2 of 12
chloroplast-encoded sequences and single-copy nuclear
genes, the ITS2 region is also bi-parentally inherited, and
hence the intra-genomic coexistence of polymorphic sequence variants can reveal the occurrence of recombination [44,45]. However, the multiple-copy nature of rDNA
renders it subject to intra-genomic variation arising from
a variety of other processes, including the generation of
paralogous somatic mutations and the degeneration of
functional genes into pseudo-genes [46]. Establishing
whether a given ribotype is taxonomically meaningful
requires the analysis of individuals rather than multigenomic samples, necessitating a single-cell approach
for unicellular dinoflagellates [20,47]. Despite its obvious
advantage in distinguishing between intra- and intergenomic sequence variation [48,49], single-cell PCR
(scPCR) has not been widely used in Symbiodinium systematics. This is primarily due to its time-consuming nature, and the difficult task of disrupting the recalcitrant
cell wall to extract the nucleic acids. A lack of suitable
methodology for isolating, extracting and sequencing
DNA from individual Symbiodinium cells has meant that
intra-genomic variation in this genus has remained virtually unexplored [50]. Fluorogenic-probe based qPCR
analysis now offers sufficient sensitivity to quantify
ITS2 variants at the sub-clade level, and has been used
to quantify polymorphic ribotypes within the individual
dinoflagellate genome through the use of a PCR preamplification step (nested qPCR; e.g. [51]).
In this study, we developed: (a) a single-cell isolation
and DNA extraction protocol for Symbiodinium; (b) a
single-cell PCR-DGGE method to screen for Symbiodinium individuals with additive ITS2 repeats; (c) a nested
PCR-qPCR assay to quantify intra-genomic ITS2 sequence polymorphisms within individual cells; and (d) a
statistical framework to identify admixture in Symbiodinium populations based on proportions of ITS2 sequence variants within the genome. The model selection
criterion developed in (d) was then employed to test
whether the P. damicornis symbiont consortium consists
of a single clonal population of symbionts featuring a
non-diagnostic polymorphism (NDP); two populations
of divergent, homogeneous symbionts; or a mixture of
genetically homogeneous symbionts and heterogeneous
cells, representing putative inter-lineage recombinants.
Methods
Study species and location
This study was carried out at the world’s southernmost
coral reef at Lord Howe Island (LHI; Australia). This isolated 14.5 km2 volcanic remnant is located around
600 km east of the Australian mainland, and some
200 km to the south of the Elizabeth and Middleton
Reefs Marine National Park Reserve. The LHI reef hosts
at least 83 species of scleractinian coral, (many of which
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
are endemic; [52]), and a correspondingly diverse and
endemic Symbiodinium assemblage [53]. The host species investigated was the widely-distributed coral Pocillopora damicornis, a thermally-sensitive but fast-growing
coral that forms a dominant component of many IndoPacific reefs (including LHI; [54]). P. damicornis is hermaphroditic and shows an unusual dual-reproductive mode,
with the majority of offspring consisting of brooded asexual larvae, complimented by the cryptic simultaneous
broadcast-spawning of sexual gametes [55]. This species
shows a predominantly sexual reproductive mode at LHI,
where it occasionally undergoes intergeneric hybridization
with Stylophora pistillata [56]. This may arise from suboptimal abiotic conditions selecting for ‘extreme’ hybrid
phenotypes and/or a low availability of conspecific gametes [57]. P. damicornis transmits symbionts vertically
from parent to offspring, and can form a symbiosis with a
wide range of genetically and physiologically distinct
Symbiodinium taxa. In Australian waters alone, P.
damicornis is found in association with S. goreauii, S.
glynni, S. trenchii, and numerous other types lacking
formal species description including C1b, C1c, C1c-ff,
C1h, C1j, C33, C33a, C42, C42a, C42b, C100, C103,
C118, C125 and C126 [4,36,53,58-62]. Of these, P.
damicornis colonies have been reported as hosting
Symbiodinium C100, C103 and C118 at LHI [53].
Sample collection and DNA isolation
Coral sampling was carried out in March 2012 at North
Bay (−31.521, 159.047) and Ned’s Beach (−31.513,
159.069), Lord Howe Island, Australia. Three P. damicornis colonies were sampled from each site by divers either
snorkelling (North Bay; depth 1–3 m) or using SCUBA
(Ned’s Beach; depth 14-16 m). Three small branch tips
(~1 cm3) were taken from each colony using diagonal pliers, and preserved in DMSO preservation buffer (20%
DMSO, 250 mM EDTA, NaCl saturated, pH 8.0; [63]).
Coral samples were stored at −20°C prior to DNA analysis. A 0.12 cm2 area of tissue was removed from the skeleton in 1.5 ml of 0.22 μm filtered seawater (FSW),
delivered at high velocity through a circular stencil. A
10 μl sub-sample was taken and centrifuged at 16,100 × g
for 5 min to pellet the Symbiodinium fraction. The supernatant was discarded and the pellet re-suspended in
100 μl DNA buffer (DNAB; 0.4 M NaCl, 50 mM EDTA,
pH 8.0). Individual cells (n = 30 from each colony) were
hand-picked under a light microscope using a heatelongated glass micro-pipette. Each cell was washed three
times in 2 μl DNAB, transferred to a 1.7 ml microcentrifuge tube with 50 mg acid-washed glass beads (710–
1180 μm; Sigma-Aldrich), and milled for 1 min at 50 Hz
(Qiagen TissueLyser LT; Qiagen, Valencia, CA, USA) to
disrupt the cell wall and release the nucleic acids. TE buffer (10 mM Tris–HCl; 1 mM EDTA; pH = 8.0) was then
Page 3 of 12
added to a final volume of 20 μl. For each colony, the extraction process was carried out with the symbiont cell
omitted (but with coral tissue homogenate included), to
ensure that only intracellular DNA contributed to the
PCR amplification signal.
End-point PCR, DGGE and DNA sequencing
Single-cell DNA template solutions generally contained
insufficient DNA for direct PCR-DGGE and qPCR analysis. The partial nr5.8S, ITS2 and partial nr28S regions
were therefore pre-amplified using a shortened endpoint PCR protocol, with the outer primers ITSintfor2
[64] and ITS2Rev2 [65]. Thermal cycling included an
initial denaturation step of 3 min at 95°C followed by
24 cycles of 15 seconds at 95°C, 15 seconds at 56°C and
10 seconds at 72°C (carried out using an Applied Biosystems Veriti thermo-cycler). Each reaction contained
10 μl of DNA template solution, 1× MyTaq PCR reaction mix (Bioline, Randolph, MA, USA), 15 pmol each
primer, and deionised sterile water to a total volume of
25 μl. A template-free control reaction was included
with each run.
Pre-amplified PCR products were diluted 1:103 (North
Bay colonies) or 1:104 (Ned’s Beach colonies) in deionised sterile water prior to PCR-DGGE and qPCR analysis (these differences were due to shortages of DNA
template solutions from the Ned’s Beach colonies,
which were used for the initial assay development and
optimization process). PCR amplification for DGGE
was carried out using the primers ITSintfor2 and
ITS2CLAMP [64]. Cycling conditions were as described
above, except that an additional 16 thermal cycles were
run (40 in total). PCR products (20 μl) were loaded on
200 × 200 × 0.75 mm, 8% denaturing polyacrylamide
gels (25-50% denaturant gradient), and run in 1 × TAE
at 150 V for 7 h at 60°C (DCode system; BioRad,
Hercules, CA, USA) alongside known ITS2 sequences
of Symbiodinium C100 and C109. Following electrophoresis, gels were stained with ethidium bromide and
viewed on a UV trans-illuminator (FirstLight UVP, San
Gabriel, CA, USA). Five representative bands at each
position were excised, milled for 1 min at 50 Hz with
50 mg glass beads and 200 μl TE buffer, and reamplified with both clamped and non-clamped primers
[64]. DGGE was carried out on clamped PCR products
to ensure a single band migrated to the identical position from where it was excised. Corresponding nonclamped products were cleaned with ExoSAP-IT (USB
Corporation, Cleveland, OH, USA), and sequenced by
the Macrogen Sequencing Service (Macrogen Inc., Seoul,
South Korea). Sequences were manually checked and
aligned in Geneious v 7.0 (Biomatters Ltd., Auckland,
New Zealand) and a BLAST search was carried out against
Symbiodinium ITS2 sequences available in GenBank.
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
Novel sequences were assigned alphanumeric ITS2 nomenclature (c.f. [24,64]) and deposited into the GenBank
database. The un-rooted statistical parsimony network of
Symbiodinium ITS2 phylotypes found within pocilloporid
corals at LHI [53] was updated in TCS v 1.21 (95%
connection limit; gaps assigned fifth character state; [66]).
qPCR analysis of Symbiodinium ITS2 ratios
For qPCR analysis, the universal primers CInnerFor
(5’-TGGCTTGTTAATTGCTTGGTTCT-3’) and CInnerRev (5’-ACCTGCATCCCAGCGGTT-3’) were developed, in addition to the custom TaqMan fluorogenic
probes C100+ and C100− (5’-TTTTACTTGAGTGA
CACCGC-3’ and 5’-CTTTACTTGAGTGACGCTGC-3’,
respectively; Life Technologies, Carlsbad, CA, USA). The
probe C100+ was designed to quantify the number of ITS2
sequences of type C100 in a given sample (denoted CC100),
while the C100− probe was developed to quantify the copynumber of all clade C ITS2 sequences other than type
C100. All primers and probes were initially checked for
specificity by conducting a BLAST search against sequences
deposited in GenBank [67]. To obtain purified DNA sequences for qPCR calibration, PCR products (types C100,
C103, C109 and C118 extracted from P. damicornis, and
C3 obtained from the Victoria University of Wellington
Symbiodinium laboratory culture collection) were cloned
using the TOPO TA kit (Life Technologies). Plasmid colonies were incubated overnight on selective LB agar plates
containing ampicillin, IPTG and XGAL (Bioline). DNA was
extracted from positive transformants, purified using a plasmid Mini-Prep kit (Life Technologies), and sequenced as
above with the M13 primer set. Plasmid DNA template
concentrations were estimated using a Pearl Nanophotometer (Implen, GmbH, Germany), diluted to approximately 10−3 ng μl−1, and five log10 serial dilutions were
constructed to generate standard curves and test the accuracy and precision of the assay. All qPCR reactions were
carried out in triplicate (standard curves) or duplicate
(template solutions) on an Applied Biosystems StepOne instrument (Life Technologies), alongside a template-free
control reaction. Each TaqMan qPCR reaction contained
4 μl template, 1× TaqMan Universal Mastermix II (Life
Technologies), 1× TaqMan fluorogenic probe (Life
Technologies), 18 pmol each primer, and deionised
sterile water to a total volume of 20 μl. Thermal cycling
conditions involved an initial 10 min, 95°C denaturation step followed by 40 cycles of 15 s at 95°C and
1 min at 60°C. Cycle threshold (Ct) values were determined
as the cycle at which the change in fluorescence was significantly different to the background level (ΔRn = 0.05; obtained using the instrument’s built-in algorithm). Ct values
below the standard curve intercept (see Additional file 1:
Table S1 and Additional file 2: Table S2) and featuring
Page 4 of 12
sufficiently low standard deviations (<0.5) were included in
the analysis.
To ensure that the TaqMan assays C100+ and C100−
detected all Symbiodinium clade C sequences present
within each sample, the total ITS2 copy number (denoted CTOTAL) in each Symbiodinium cell from the
North Bay colonies was also estimated using SYBR
qPCR analysis. Reactions were carried out as above, except Power SYBR Green Mastermix (Life Technologies)
was used in place of TaqMan Universal Mastermix II,
fluorogenic probes were omitted, and Ct values were
generated using the ΔRn threshold value of 0.3. A melt
curve (temperature elevation from 60°C to 95°C in 0.3°C
increments each of 15 s duration) was included at the
end of each run to ensure that only target sequences
were amplified. Template solutions yielding Ct values
below the standard curve intercept and melting temperatures (Tm) within 1°C of plasmid Tm values were included in the analysis. The ITS2 copy number within
each cell (CTOTAL; as determined from SYBR qPCR analysis) was compared to the sum of those given by the
C100+ and C100− TaqMan assays using linear regression
(parameters constrained; intercept = 0, slope = 1). Finally,
a mixture test was carried out to assess the ability of the
TaqMan qPCR assay to predict the proportion of total
Symbiodinium clade C ITS2 copies that were of type
C100 (CC100:CTOTAL ratio). Eight mixtures were constructed from plasmid C100 and C109 DNA template
solutions (diluted to approximately 200 ITS2 copies μl−1;
CC100:CTOTAL ratios = 0, 0.02, 0.10, 0.4, 0.6, 0.9, 0.98 and
1; see Additional file 3: Table S3 for Ct values) and qPCR
reactions were carried out in duplicate as above. The
ability of the combined TaqMan assay to predict CTOTAL
and CC100:CTOTAL was assessed using linear regression
(parameters constrained; intercept = 0, slope = 1).
Statistical analysis
To assess the relationship between the total ITS2 copy
number and the proportion of copies that were of type
C100, a non-linear regression curve (second order polynomial) was fitted to the bivariate CC100:CTOTAL versus
CTOTAL data in Sigmaplot v11.0 (Systat, Richmond, CA,
USA). Values of CC100:CTOTAL were arcsin transformed
and compared between colonies (Colony) and between
branches within colonies (Branch(Colony)) using nested
ANOVA (lm function in R; [68]). Three competing hypotheses were evaluated to explain the ITS2 sequence
variation within and between the symbionts of P. damicornis: (H0) colonies host a single population of genetically heterogeneous symbionts, versus (H1) colonies host
two populations of genetically distinct, homogeneous
symbionts, versus (H2) colonies host distinct populations
of genetically homogeneous and heterogeneous symbionts, consistent with the occurrence of recombination
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
Page 5 of 12
(Figure 1a). The proportion of each ITS2 type in the
genome of a heterogeneous cell may deviate from codominance (50% each ITS2 type) if recombinants backcross to
one or both parental populations (i.e. introgression;
Figure 1b). The frequency distribution of CC100:CTOTAL
within a coral colony (X) is expressed in model form as:
H 0 : X eBetaðα; βÞ; α > 1; β > 1
H 1 : X eBetaðα; βÞ; α < 1; β < 1
H 2 : X eπBetaðα1 ; β1 Þ þ ð1–π ÞBetaðα2 ; β2 Þ; 0 < π < 1
where α and β are the shape parameters of the beta
function, and π denotes the proportion of symbionts belonging to each component of the mixture model. Mixed
beta functions were fitted to the CC100:CTOTAL frequency
distributions of each coral colony, and maximum likelihood parameter values were solved using the optim
function in R (L-BFGS-B method; [68]). A range of starting parameter values was used at each optimization stage
to ensure that a universal log-likelihood maximum was
reached. Hypothesis evaluation was based on weighted
AICc values (wi), with those above 0.90 considered to provide unambiguous support for a candidate model [69,70].
(a)
H0
H1
(b)
H2
F1 hybrid
F1 hybrid
F2 backcross
F2 backcross
Fn backcross
Figure 1 Conflicting origins of intra-genomic variation in
Symbiodinium. Red and green colourations represent divergent ITS2
sequences, with monocoloured cells featuring homogeneous ITS2
arrays and bicoloured cells hosting polymorphic ribotypes. Schematics
show (a) competing hypotheses of sequence homology within P.
damicornis-associated Symbiodinium, with a single clonal population
hosting a NDP under H0, two genetically isolated populations under H1
and inter-lineage recombination under H2; and; and (b) introgression,
with fitness disparities between F1 and later generations (shown in a
size gradient). Many backcross generations (n) may occur before an
increase in fitness is realized. Genetic isolation occurs when one or
more classes suffer from insurmountably low mean fitness.
Results
DGGE and DNA sequencing
The excision and sequencing of DGGE bands revealed
that all six P. damicornis colonies hosted Symbiodinium
ITS2 types C100 (GenBank accession number HM222433;
[53]) and C109 (GenBank accession number KJ530690;
novel sequence). No other Symbiodinium sequences were
detected, including the rare types C103 and C118 previously identified from P. damicornis at LHI [53]. While the
low resolution and sensitivity of DGGE may have simply
precluded their detection, this is unlikely given that this
was the same method used in [53]. Alternatively, the absence of C103 and C118 may be explained by differences
in host-identification between studies. For example, two
ambiguous colonies omitted from the present study that
appeared to be the P. damicornis × Stylophora pistillata
hybrids described in [56] were later found to exclusively
host Symbiodinium C118 (S.P. Wilkinson, unpublished
data). Although two divergent ITS2 sequences were retrieved, three distinct DGGE band profiles were observed
among the 180 individual cells analysed. These corresponded to Symbiodinium cells featuring a homogeneous
C109 array, those featuring a near-homogeneous C100
array (with trace copies of C109; hereafter referred to as
homogeneous C100), and those with a co-dominant mixture of both ITS2 types (Figure 2). Four of the six colonies
analysed hosted a consortium of Symbiodinium cells that
included all three profiles (two colonies from each site),
while the remaining two colonies hosted only homogeneous C100 symbionts and those producing the heterogeneous band-pattern (Figure 2a). No amplification signal
was detected from template-free controls or the extractions with symbiont cells omitted, indicating an absence
of extracellular DNA contamination.
qPCR estimation of intra-genomic ITS2 ratios
The universal primers CInnerFor and CInnerRev were
identically matched to conserved regions within the
ITS2 of Symbiodinium C100 and C109. These primers
also share identical sequences or single-base pair mismatches with nearly all clade C sequences currently
available in the GenBank database, including those
found within the corals of LHI [53]. A sequence BLAST
analysis of the target probe C100+ revealed a high specificity for Symbiodinium C100, with at least two nucleotide substitutions differentiating it from the majority of
other clade C sequences in GenBank (positioned 16 and
18 base pairs from the 5’ end of the probe). The cytosine
at the 5’ end of the probe C100− is mismatched to C100,
C109 and the majority of other clade C Symbiodinium
types (including the ancestral types C1 and C3). This
mismatch had no effect on the reaction efficiency when
tested on ITS2 types C109 and C103 (95% < E < 100%;
see Additional file 1: Table S1 and Additional file 2: Table
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
(b)
< 0.01
0.45
0.63
0.77
0.86
single cells
0.95
0
plasmid
CC100:CTOTAL = 1
(a)
Page 6 of 12
C118
C100
C103
C1
C109
1 bp
C3
C3hh
C100
C109
C1bb
C3n
C3gg
Figure 2 Sequence variation among pocilloporid-associated Symbiodinium at LHI. ITS2 sequence variation between and within the Symbiodinium
genome is shown by: (a) DGGE profiles of individual symbionts from P. damicornis colonies at Lord Howe Island, featuring a range of CC100:CTOTAL ratios
(alongside plasmid-purified C100 and C109 DNA); and (b) an un-rooted statistical parsimony network showing the phylogenetic relationships between
derived pocilloporid-associated Symbiodinium types found at Lord Howe Island (ellipses) and the ancestral C3 root (rectangle; modified from [53]). Small
circles in (b) represent hypothetical intermediate sequences, each distinguished from its neighbour by a single nucleotide substitution or gap.
P. damicornis-associated types are shown in yellow, while those found in association with Stylophora pistillata and Seriatopora hystrix are shown
in orange and green, respectively.
S2); however it served to prevent cross-hybridization with
the C100 sequence. With this exception, C100− shared an
identical sequence to most clade C Symbiodinium types
available in GenBank, including the ancestral types C1, C3
and all derived types found in association with P. damicornis at LHI [53]. Standard curve analysis of both TaqMan
assays revealed acceptable reaction efficiencies when
matched to their respective target sequences (C100+ to
C100; C100− to both C109 and C103; 95% < E < 100% and
R2 > 0.99 in all cases; see Additional file 1: Table S1 and
Additional file 2: Table S2). qPCR analysis of known plasmid DNA mixtures yielded high accuracy and precision in
estimating CC100:CTOTAL (constrained linear regression;
R2 = 0.998; Additional file 3: Table S3, Additional file 4:
Figure S1a) and an absence of cross-hybridization.
TaqMan qPCR-generated C TOTAL values within each
Symbiodinium cell were highly correlated with, and
not significantly different from those obtained from
the SYBR qPCR assay (constrained linear regression;
R2 = 0.978; Additional file 4: Figure S1b), indicating a
negligible incidence of clade C ITS2 types other than
those detected by C100+ and C100−. SYBR qPCR melt
curve analysis showed no Tm differences between plasmid C100 and C109, and all single-cell templates
yielded single Tm peaks within 1°C of the plasmidgenerated values.
Within-cell ITS2 copy numbers (CTOTAL) ranged from
less than 500 to over 30,000, and CC100:CTOTAL ratios
ranged between 0 and 0.987 (Figure 3; see Additional file
5: Table S4, Additional file 6: Table S5, Additional file 7:
Table S6, Additional file 8: Table S7, Additional file 9:
Table S8 and Additional file 10: Table S9 for Ct values).
The remaining ITS2 copies appeared to be primarily of
type C109, since this was the only other sequence
detected in the DGGE analysis. DGGE band intensities
generally reflected qPCR-generated CTOTAL values, and
in cases where both C100- and C109-diagnostic bands
were present, their relative intensity gave a qualitative
indication of CC100:CTOTAL. However, the C109 band
was generally very faint in cells featuring CC100:CTOTAL
ratios greater than 0.75, and universally undetectable in
those above 0.85 (Figure 2a; see Additional file 5: Table
S4, Additional file 6: Table S5, Additional file 7: Table
S6, Additional file 8: Table S7, Additional file 9: Table S8
and Additional file 10: Table S9). A significant nonlinear correlation between CC100:CTOTAL and CTOTAL revealed that ITS2 copy numbers were higher on average
in genetically homogeneous C100 cells than in either
the heterogeneous C100/C109 cells or the homogeneous
C109 cells (non-linear regression, p < 0.027; R2 = 0.15;
Figure 3). Within-cell CC100:CTOTAL ratios did not differ
between branches within colonies, but varied between colonies (nested ANOVA, p = 0.82 and < 0.01 for Branch
(Colony) and Colony effects, respectively; Table 1).
The application and evaluation of competing beta
models based on CC100:CTOTAL ratios revealed the presence
of multiple symbiont clusters in all six colonies. In all cases,
the two-component beta mixture model representative of
H2 provided the best fit of the candidate models (wi > 0.90
for all colonies; Table 2). Three modes were present in
colonies a, b, d and e, representing clusters of genetically homogeneous C100 cells, homogeneous C109 cells,
and heterogeneous C100/C109 cells. Two modes were
detected in colonies c and f, representing coexisting
populations of homogeneous C100 cells and heterogeneous C100/C109 cells (Figure 4). The proportion of
genetically heterogeneous symbiont cells in the consortium ranged from 7% in colony c to 88.5% in colony a.
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
Page 7 of 12
Method development
Figure 3 Variation in ITS2 copy numbers within Symbiodinium
cells. A non-linear relationship existed between the proportion of ITS2
copies of type C100 (CC100:CTOTAL) and the total number of ITS2 copies
within the cell (CTOTAL). Homogeneous C100 cells hosted significantly
more ITS2 copies than the genetically heterogeneous cells, or those
featuring a homogeneous C109 array (second order polynomial
regression, p = 0.027).
Discussion
Assessing the incidence of recombination between divergent Symbiodinium lineages is made difficult by their apparent haplontic life cycle, a lack of amenability to
culture in many types (particularly in clade C Symbiodinium), and the paucity of high-resolution single-copy
genetic markers. This study attempts to circumvent
these obstacles by developing protocols to isolate and
extract DNA from individual Symbiodinium cells, establish and quantify the dominant ribotype(s) within each
genome, and test competing hypotheses explaining the
observed pattern of intra-genomic variation. Using these
techniques, a population of putative inter-lineage recombinants is identified inhabiting the reef building coral
Pocillopora damicornis at the isolated, high-latitude reef
of Lord Howe Island, Australia.
Table 1 Nested ANOVA output for intra-genomic
variation in ITS2 ratios
Source of variation
df
SS
MS
F
P
Between colonies
5
4.07
0.81
24.23
0.001
Between branches within colonies
12
0.40
0.03
0.61
0.82
Error
162
8.87
0.05
The model design used in the nested ANOVA analysis was CC100:CTOTAL ~
Colony + Branch(Colony). CC100:CTOTAL ratios were arcsin transformed prior to
analysis. Branches within colonies were pooled for subsequent mixture
model fitting.
The single-cell isolation and extraction method described
here facilitated the rapid preparation of individual Symbiodinium cells prior to PCR (around 20 per hour), with the
potential to be further improved with the application of
flow-cytometry and fluorescence activated cell sorting
(FACS). The protocol also showed good efficiency, with
around 85% of isolated cells undergoing successful PCR
amplification. The downstream application of DGGE
and DNA sequencing successfully revealed the dominant ribotype(s) within individual cells, providing a reliable assessment of inter-genomic ITS2 diversity within
the P. damicornis symbiont consortium. Used in conjunction with plasmid cloning, this method could be
used to evaluate levels of intra-genomic variation in
other genetic markers, providing an important assessment of their phylogenetic utility.
The qPCR assay developed in this study offers sufficient sensitivity to quantify ITS2 ratios at the sub-clade
level. This represents a significant improvement in resolution from earlier clade-level assays [71-76], since the
sub-clade presents a more ecologically-relevant taxonomic
unit [19]. This assay is also the first to quantify polymorphic rDNA sequences within individual Symbiodinium cells, and the second to do so in dinoflagellates (see
also [51]). This provides an important insight into the
level of ITS2 variation within the Symbiodinium genome,
underscoring concerns about its utility in establishing diversity estimates [46], and its suitability for quantifying the
dynamics of mixed infections [74]. In particular, substantial differences in rDNA copy numbers observed between
Symbiodinium types C100 and C109 highlight the perils of
using ITS2-qPCR to estimate abundance ratios of coexisting symbionts without single-cell validation. Finally, the
statistical methodology developed here can identify potential admixture in symbiont populations based on intragenomic ITS2 ratios. Conflicting hypotheses of one, two
and three coexisting populations were formulated, corresponding to the existence of a single symbiont clone harbouring a non-diagnostic polymorphism (NDP), the
coexistence of two ‘pure’ (homogeneous) ribotypes, and
mixed populations of genetically homogeneous and heterogeneous Symbiodinium cells, respectively. The model
consistent with the latter hypothesis received unambiguous statistical support in all six P. damicornis colonies
analysed. However, the model selection approach relies on
forming a set of candidate models that are representative
of the biological processes under investigation [70]. While
the mixture model representing H2 is consistent with a
population of recombinant genotypes coexisting with parental populations (progenitors), it cannot explicitly prove
this scenario. This is because a similar pattern could arise
from the incomplete concerted evolution of ancestral
polymorphisms (ICEAP).
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
Page 8 of 12
Table 2 Summary of optimized beta mixture models
Colony ID
Sample site
Best-fit
hypothesis
Model equation
Proportion of
heterogeneous cells
Akaike weight (wi)
a
North Bay
H2
X ~ 0.13 × Beta (0.57, 0.53) + 0.87 × Beta (26.08, 10.89)
0.885
> 0.99
b
North Bay
H2
X ~ 0.07 × Beta (3.48, 66.21) + 0.93 × Beta (10.99, 2.89)
0.816
> 0.99
c
North Bay
H2
X ~ 0.07 × Beta (13.97, 10.32) + 0.93 × Beta (57.10, 3.12)
0.007
> 0.99
d
Ned’s Beach
H2
X ~ 0.33 × Beta (0.71, 0.57) + 0.67 × Beta (108.55, 68.91)
0.670
> 0.99
e
Ned’s Beach
H2
X ~ 0.51 × Beta (16.58, 4.28) + 0.49 × Beta (114.8, 2.32)
0.512
0.96
f
Ned’s Beach
H2
X ~ 0.55 × Beta (0.90, 0.34) + 0.45 × Beta (19.40, 10.63)
0.444
0.93
Model support is indicated by Akaike weights (wi), representing the conditional probability that a particular model provides the best fit of all candidate models
(i.e. H0, H1 and H2). These give unambiguous support for a candidate if > 0.9 [69,70].
Recombination or incomplete concerted evolution of
ancestral polymorphisms?
The existence of both C100 and C109 ribotypes in the
homogeneous condition affirms their status as diagnostic
of separate Symbiodinium sub-clades (i.e. neither sequence represents a degenerating pseudo-gene). Furthermore, these two ribotypes differ at five variable nucleotide
sites in the ITS2 region (2% divergence), while NDPs typically feature a single nucleotide substitution or insertion/
deletion (indel) that distinguishes them from the dominant sequence variant [19,36]. However, if both ribotypes
were present within the genome of the most recent common ancestor of Symbiodinium C100 and C109, processes
of concerted evolution may not have had sufficient time
to homogenize the rDNA arrays of both taxa. Hence copies of the ribotype that is now diagnostic of the sister
taxon may remain in the genome of one or both lineages.
The Symbiodinium genome routinely hosts a diverse assemblage of ITS2 sequences [46], and several putative
cases of ICEAP appear in the literature. For example, the
ITS2 sequence diagnostic of Symbiodinium glynni (type
D1) also occurs within the genome of S. trenchii (type
D1a), with the incomplete displacement of a vestigial polymorphism invoked to explain their intra-genomic coexistence [46]. However, several features of the data presented
here suggest that an alternative explanation of recombination is feasible. First, the C100 and C109 sequences coalesce at the ancestral type C3, as opposed to either
representing an intermediate evolutionary step toward the
other (e.g. C103 and C118 in P. damicornis and C3hh and
C3n in Seriatopora hystrix; see Figure 2b). If concerted
evolution has not had sufficient time to homogenize all
C109 rDNA repeats in the C100 genome, then vestigial
copies of the intermediate C3 sequence would also likely
persist as a non-dominant intra-genomic variant. Rather,
the C3 sequence was not detected in any of the cells
analysed, despite its characteristic DGGE band pattern
(see supplementary material in [53]). Second, concerted
evolutionary processes rapidly homogenize intra-genomic
co-dominance, either completely displacing a nondominant polymorphism or leaving only background traces
[44,77,78]. This is inconsistent with the similar proportional abundance of ITS2 polymorphisms within many of
the genetically heterogeneous cells observed here, with
more than a third of all symbionts featuring CC100:CTOTAL
ratios of between 0.25 and 0.75. Finally, frequency ‘dips’
along the CC100:CTOTAL spectrum depict a degree of genetic
isolation between genetically heterogeneous Symbiodinium
cells and either of the ‘pure’ genotypes (i.e. homogeneous
C100 and C109 cells), consistent with the substantial fitness
loss often experienced by F2 and later-generation backcross
genotypes (as a result of processes such as ‘hybrid breakdown’; see [79,80]).
While recombination represents a plausible explanation
for the intra-genomic codominance of the C100 and C109
ribotypes, there remains a possibility that this pattern resulted from ICEAP. Addressing this question will likely require a significant investment of resources, including the
development of a suite of single-copy markers, the generation of isoclonal cultures or the application of whole genome amplification (WGA; in order to facilitate multilocus genotyping analysis on individual cells), and/or continued attempts to induce the sexual life cycle, both within
and between cultured Symbiodinium lineages. Another area
requiring investigation is the morphological, physiological
and ecological characterization of putative Symbiodinium
recombinants. Concerted evolution operates via a series of
stochastic processes that occur independently of natural
selection [81]. By contrast, recombination between lineages
is often accompanied by drastic changes in morphology,
performance and fitness [79,82-84], even involving diversification into new habitats [85]. Investigating the form, function, distribution and ecology of genetically heterogeneous
Symbiodinium cells may therefore provide further insight
into the incidence and potential evolutionary effects of
recombination within and between Symbiodinium lineages.
Background symbiont populations
The results of this study indicate that at least three ITS2
genotypes can coexist within the symbiont consortium
of P. damicornis (C100, C100/C109 and C109). While
homogeneous Symbiodinium C109 cells were only ever
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
Page 9 of 12
Figure 4 Frequency distributions of intra-genomic ITS2 ratios in coral colonies. Colonies a-c were sampled from North Bay (1-3 m), and
colonies d-f from Ned’s Beach (14-18 m). Vertical bars represent the percentage of symbiont cells within each CC100: CTOTAL category (n = 30 cells
for each colony). Overlying probability density functions are optimized two-component beta mixtures (see Table 2 for parameter estimates).
detected at background levels (constituting less than 7%
of the symbiont population), the biological relevance of
this population may extend well beyond providing a presumably minor contribution to the overall productivity
of the symbiosis. Genetically heterogeneous Symbiodinium cells outnumbered ‘pure’ genotypes in more than
half of the colonies sampled, suggesting that rare sexual
reproduction events between C100 and C109 may facilitate asexual proliferation of the F1 generation, with
potentially important functional implications for the
coral colony. The evolutionary contribution of rare Symbiodinium types may be more important still, if recombinants create a ‘bridge’ for the migration of genetic
material to the dominant lineage (i.e. introgression; see
Figure 1b). A small number of genetically heterogeneous
symbionts featured CC100:CTOTAL ratios near 0.75, and
thus potentially represent F1 × C100 backcross genotypes. However, this pattern could equally have arisen
Wilkinson et al. BMC Evolutionary Biology (2015) 15:46
from ICEAP, differential rDNA inheritance in the F1
generation (arising from dissimilar copy-numbers between parent taxa; e.g. [51]), or even concerted evolution
acting to homogenize rDNA variability in the recombinant genome (e.g. [77]). Establishing the incidence of
introgression would initially require the identification of
individual F1- and backcross classes. This in turn requires the genotyping of a large number of individuals,
and the analysis of at least 13–50 ancestry-informative
loci per individual [86,87]. This study was not sufficiently resourced to carry out such a comprehensive
task; however it does serve to highlight the perils of dismissing symbionts that persist in low abundance as
biologically-irrelevant or simply representing surface
contamination.
Conclusion
While the results presented in this study do not provide
unequivocal evidence of recombination between divergent
Symbiodinium lineages, they provide an initial ‘proof of
principle’ for its occurrence. In doing so, this study draws
attention to the important evolutionary implications that
may accompany the generation of new genetic diversity in
Symbiodinium, including the potential for rapid symbiont
adaptation through introgression. Progress in this area has
been hindered by a lack of available methodology, an obstacle that is addressed here through the development of
new molecular and statistical methods focused on the individual Symbiodinium cell. Additional development of
this research may help to characterize and predict the evolutionary response of the coral-algal symbiosis to the
many anthropogenic impacts currently threatening the
world’s coral reefs.
Data accessibility
Amino acid sequence data is deposited in GenBank (accession number KJ530690)
Quantitative PCR (qPCR) cycling threshold values and
model parameters accompany the manuscript as supplemental information.
Additional files
Additional file 1: Table S1. Standard curve analysis for nested qPCR
(North Bay colonies).
Additional file 2: Table S2. Standard curve analysis for nested qPCR
(Ned’s Beach colonies).
Additional file 3: Table S3. Assay validation for TaqMan nested qPCR.
Additional file 4: Figure S1. Single-cell qPCR assay validation.
Additional file 5: Table S4. Mean Ct values for individual Symbiodinium
cells (colony a).
Additional file 6: Table S5. Mean Ct values for individual Symbiodinium
cells (colony b).
Page 10 of 12
Additional file 7: Table S6. Mean Ct values for individual Symbiodinium
cells (colony c).
Additional file 8: Table S7. Mean Ct values for individual Symbiodinium
cells (colony d).
Additional file 9: Table S8. Mean Ct values for individual Symbiodinium
cells (colony e).
Additional file 10: Table S9. Mean Ct values for individual Symbiodinium
cells (colony f).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SPW, PLF & SKD conceived and designed the project. SPW collected samples,
performed assays and analyzed the data. SPW, PLF, MJHvO & SKD wrote the
paper. All authors read and approved the final manuscript.
Acknowledgements
SPW was supported by a VUW Vice Chancellor’s Strategic PhD Scholarship, JL
Stewart Scholarship, and a Royal Society Marsden Fund Grant awarded to
SKD and PLF (contract number VUW0902). Corals were collected under New
South Wales Department of Primary Industries permit number P10/0042-1.1.
The work was made possible with the help of the Lord Howe Island Marine
Parks Authority (S. Gudge, J. Maher & I. Kerr) and the efforts of Lord Howe
Island residents (I. Fitzgerald, R. Moran & T. Solomon). We thank G. Zuccarello,
X. Pochon, S. Santos, N. Phillips and J. Kennington for critical discussion and
manuscript comments, S. Pledger for statistical advice, S. Pontasch for help
with sample collection, and the anonymous reviewers whose suggestions
substantially improved the quality of this manuscript.
Author details
1
School of Biological Sciences, Victoria University of Wellington, Kelburn
Parade, Wellington 6012, New Zealand. 2Australian Institute of Marine
Science, PMB No. 3, Townsville, QL 4810, Australia.
Received: 1 December 2014 Accepted: 24 February 2015
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